Re esseaarrc chh iSSuubbcoommmitttteeee R eessoouurrcce ... Carnivores Jason Herrick jherrick@

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  • RReesseeaarrcchh SSuubbccoommmmiitttteeee

    RReessoouurrccee MMaannuuaall wwwwww..iieettss..oorrgg CANDES Research Subcommittee (for additional copies)

    AA CCoommppiillaattiioonn ooff SSttaannddaarrdd OOppeerraattiinngg PPrroocceedduurreess ffoorr

    EEmmbbrryyoo TTrraannssffeerr aanndd RReellaatteedd TTeecchhnnoollooggiieess ffoorr CCAANNDDEESS

    Updated June 2011

    To submit new protocols, contact the relevant Taxon Leader or Subcommittee Chairmen

    (see p. 4 for contact information)


    I. Carnivores (Taxon Leader: Jason Herrick)

    1. Gamete/Embryo Collection a. Large Felid Semen Collection by Rectal

    Probe Electrostimulation (N.M. Loskutoff) 5

    b. In Vitro Fertilization and Embryo Transfer in Felids (C.E. Pope) 9

    2. Cryopreservation

    a. Large Felid Semen Cryopreservation (N.M. Loskutoff) 26

    b. Canid Semen Cryopreservation (W. Farstad) 30

    3. Endocrinology

    a. ELISA Protocol: Measuring Anti-Gonadotropin Immunoglobulin Titers (W.F. Swanson) 33

    II. Ungulates (Taxon Leader: Rebecca L. Krisher)

    1. Gamete/Embryo Collection

    a. Wild Cattle, Buffalo and Bison Semen Collection by Rectal Probe Electrostimulation (N.M. Loskutoff) 36

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    2. Embryo Transfer

    a. Superovulation and Embryo Transfer in the Dromedary Camel (J.A. Skidmore) 40

    3. In Vitro Fertilization/Culture

    a. Bovine IVM/IVF/IVC Protocol (R.L. Krisher) 47

    4. Cryopreservation

    a. Wild Cattle, Buffalo and Bison Semen Cryopreservation (N.M. Loskutoff) 55

    b. Generalized Procedures for Harvesting Epididymal Sperm from Ruminants Post-Mortem for Cryobanking (N.M. Loskutoff) 58

    III. Birds (Taxon Leader: Juan M. Blanco)

    1. Artificial Insemination

    a. Semen Collection and Artificial Insemination in Cranes (R. Lastovica & N.M. Loskutoff) 66

    IV. Amphibians and Reptiles (Taxon Leader: Andrew J. Kouba)

    1. Gamete/Embryo Collection

    a. Semen Collection in Snakes (E. Viera & E.G. Crichton) 71

    b. Artificial Insemination in Snakes (K. Mattson & N.M. Loskutoff) 73

    V. Non-Human Primates (Taxon Leader: Justine K. O’Brien)

    1. In Vitro Fertilization/Culture

    a. Human sperm – Oocyte Interaction Tests (D.Y. Liu & H.W.G. Baker) 74

    2. Cryopreservation

    a. Gorilla Sperm Cryopreservation Protocol: Non-Egg Yolk Diluent (J.K. O‘Brien) 77 b. Gorilla Sperm Cryopreservation Protocol: Test Yolk Buffer Diluent (J.K. O‘Brien) 79 c. Great Ape Semen Cryopreservation Protocols: Egg Yolk-Based Cryodiluents (T.R. Bowsher & N.M. Loskutoff) 81

    3. Endocrinology

    a. Fecal Extraction Protocol for Hormone Analysis (A.C. Bellem and N. Wielebnowski) 86

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    VI. Marsupials (Taxon Leader: Monique C.J. Paris)

    1. Gamete/Embryo Collection

    a. Collection and Evaluation of Brushtail Possum Semen/Sperm (F. Molinia) 87

    b. Superovulation of Female Brushtail Possums (F. Molinia) 93

    c. Superovulation of Womat Species (M.C.J. Paris & G. Druery ) 95

    d. Superovulation, Oocyte Collection, In Vitro Maturation, Semen Collection and Intracytoplasmic Sperm Injection in the Tammar

    Wallaby (G. Magarey) 98

    2. Endocrinology

    a. Estrous Cycle Monitoring n Wombat Species (M.C.J. Paris & G. Finlayson) 106

    b. Wombat Faecal Progesterone Metabolite Analysis (M.C.J. Paris & F. Schwarzenberger) 109

    c. Luteinizing hormone radioimmunoassay (M.C.J. Paris) 114

    3. Cryopreservation

    a. Ovarian Tissue Cryopreservation (J. Shaw) 117 VII. Fish (Taxon Leader: Boris Dzyuba)

    1. Cryopreservation

    a. Fish Semen Cryopreservation. Patterns for Common Carp, Cyprinus carpio, Barbel Sturgeon, Acipenser nudiventris, and Haarder, Mugil soiuy (B. Dzyuba) 126

    2. Other a. Measurement Techniques for Fish Larvae Morphometric

    Parameters on Early Stages of Postnatal Growth (B. Dzyuba, K.J.W. Van Look, N. Satake & W.V. Holt) 132

    VIII. Rodents (Taxon Leader: Marina F. Ponzio)

    1. Semen Collection and Cryopreservation

    a. Semen Collection, Evaluation and Cryopreservation in the Chinchilla, Chinchilla lanigera (M. Ponzio) …………………………... 133

    VIII. General

    1. Endocrinology

    a. Cortisol Enzyme Immunoassay Protocol (A.C. Bellem, C. Munro and N. Wielebnowski) 137

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    IIEETTSS CCAANNDDEESS RReesseeaarrcchh SSuubbccoommmmiitttteeee E-Mail Addresses of Resource Manual Taxon Leaders

    Taxon Taxon Leader E-Mail Address

    Carnivores Jason Herrick

    Ungulates Rebecca L. Krisher

    Birds Juan Manual Blanco

    Amphibians/Reptiles Andrew Kouba

    Non-Human Primates Justine K. O‘Brien

    Invertebrates Anita Collins

    Marsupials Monique C.J. Paris

    Fish Boris Dzyuba

    Marine Mammals Todd Robeck

    Rodents Marina F. Ponzio

    Other Taxa Suggestions Requested

    The IETS CANDES Research Subcommittee Resource Manual includes standard operating

    procedures (defined here as protocols) referring to all embryo transfer and related technologies

    applicable to companion animals, non-domestic and endangered species.

    New information and updates will be posted in March and September annually. Authors may

    send copies of their protocols for posting on the IETS CANDES web page to the relevant Taxon Leader

    above. Each new submission must contain contact details (preferably an e-mail address) for queries

    concerning the protocols and they must include citations of supporting manuscripts and/or data

    demonstrating application of the procedures. Protocols are specifically requested for the following


    1. Gamete/Embryo Collection 2. Embryo Transfer 3. Artificial Insemination 4. In Vitro Fertilization/Culture 5. Cryopreservation 6. Endocrinology

    Taxon Leaders are needed at this time for ―Birds‖ and ―Marine Mammals‖ and requests are

    being accepted for other taxa to be included in the Resource Manual. Queries and requests should be

    sent to the IETS CANDES Research Subcommittee Co-Chairmen: Rebecca L. Krisher (E-Mail: or Monique C. J. Paris (E-Mail: Taxon Leaders

    should have some background knowledge or experience in embryo transfer and related technologies for

    their specific taxon, and are responsible for recruiting participation from colleagues to submit relevant


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    Large Felid Semen Collection by Rectal Probe

    Electrostimulation (Electroejaculation)

    N.M. Loskutoff

    Center for Conservation & Research, Omaha‘s Henry Doorly Zoo

    September 2003

    Contact information for further details:

    Naida Loskutoff at:

    Publications using this protocol:

    1) Crichton, E.G., E. Bedows, A.K. Miller-Lindhom, D.M. Baldwin, D.L. Armstrong, L.H. Graham, J. Ford, J.O. Gjorret, P. Hyttel, C.E. Pope, G. Vajta and N.M. Loskutoff (2003) The

    efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval

    and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

    Biology of Reproduction 68: 105-113. 2) Gjorret, J.O., Crichton, E.G., Loskutoff, N.M., Armstrong, D.L. and Hyttel, P. Oocyte

    maturation, fertilization and early embryonic development in vitro in the Siberian tiger (Panthera tigris altaica). Molecular Reproduction and Development 63:79-88, 2002.

    3) Nelson, K.L., E.G. Crichton, L. Doty, D. Volenec, J.M. Finnegan, R.G. Morato, C.E. Pope, B.L. Dresser, D.L. Armstrong and N.M. Loskutoff (1999) Heterologous and homologous fertilizing

    capacity of cryopreserved felid sperm: a model for endangered species. Theriogenology 51:290,


    4) Donoghue, A., A. Byers, L. Johnston, D. Armstrong, and D. Wildt. Timing of ovulation after gonadotrophin induction and its importance to successful intrauterine insemination in the tiger

    (Panthera tigris). Journal of Reproduction and Fertility, 107: pp 53-58. 1996

    Materials Needed:

    Rectal probe (see Fig. 1), sand paper

    Electrostimulator (e.g., Platz or Beltz models – for contact details,

    see Technology Subcommittee Resources section)

    Electrical extension cord